Improvement of plant varieties through genetic transformation has become increasingly important for modern plant breeding. Genes of potential commercial interest, such as genes conferring to a plant traits of disease resistance, insect resistance or improved quality, may be incorporated into crop species through various gene transfer technologies.
The development of an efficient transformation system is necessary for the analysis of gene expression in plants. The requirements for such a system include a proper target plant tissue that will allow efficient plant regeneration, a gene delivery vehicle that delivers foreign DNA efficiently into the target plant cells, and an effective method for selecting transformed cells. In genetic transformation of dicotyledonous species, for example, transformation systems utilizing the bacterium Agrobacterium tumefaciens have been frequently used as vehicles for gene delivery. The preferred target tissues for Agrobacterium-mediated transformation presently include cotyledons, leaf tissues, and hypocotyls. High velocity microprojectile bombardment offers an alternative method for gene delivery into plants.
Although genetic transformation and subsequent regeneration is largely a matter of routine nowadays for many plants species, several commercially significant crops such as sugar beet, squash, sunflower, soybean, and cotton have remained recalcitrant to transformation by most of the numerous methods that are available.
Beta vulgaris (which includes sugar beet, fodder beet, table beet and Swiss chard) is one example where, despite transient expression in some cells and occasional success with specific genotypes, no simple routine method is available for the production of transgenic plants. The recalcitrance to transformation of sugar beet protoplasts is well-documented. See, for example, International Patent Application No. WO 91/13159 and K. D'Halluin et. al., Bio/Technology, 10 309-314 (1992)). Regarding Agrobacterium-mediated gene transfer, “the sugarbeet's recalcitrance is renowned,” and “most of the techniques reported for the production of transgenic sugarbeet plants require the expert skill of laboratories that developed them and [has] proved not easily reproducible by others.” F. A. Krens et al., Plant Sci. 116, 97-106 (1996), citing M. C. Elliot et al., in Physiology and Biochemistry of Cytokinins in Plants, pages 329-334 (SPB Publishing, The Hague, 1992); K. Lindsey et al., J. Exp. Bot. 41, 529-536 (1990); and D'Halluin et al., supra. The Krens et al. paper states further that “the method of choice . . . is one using cotyledons as the explant system, which has only been described superficially.” Krens et al., supra, citing J. E. Fry et al., Abstract # 384 in Molecular Biology of Plant Growth and Development, Third International Congress of the International Society for Plant Molecular Biology (R. B. Hallick, editor, Tuscon, Ariz., USA 1991). However, even this method is not highly efficient; as an example, one researcher reported obtaining only 21 transgenic shoots, including several chimeras, from 15,000 inoculated explants. U. Sander, Transformation von Beta vulgaris L., (Ph.D. Thesis, University of Hanover, Germany 1994). The Krens et al. paper itself reports a 0.9% transformation frequency in sugarbeets using a cotyledonary node and kanamycin selection technique. Krens et al., supra, at page 103.
There are brief descriptions in the literature relating to callus production from the epidermal cells of sugar beet (see Kotowska et al., Bull. of the Polish Acad. Sci. 32, 11-12 (1984); Kotowska, Beitr. Biol. Pflanz. 67, 209-223 (1992)), and several reports describing adventitious bud production on the epidermis of sugar beet petioles. See e.g., Harms et. al., Plant Cell Tissue Organ Culture 2, 93-102 (1983); Schneider et al., Biochem. Physiol. Pflanz. 182, 485-490 (1987)). However, these reports present no evidence that transformed sugar beet plants can be regenerated using these methods.
Sugarbeet transformation using sugarbeet protoplasts (through stomata guard cells) has been reported. See, e.g., R. Hall et al., Nature Biotechnology 14, 1133-1138 (1996); R. Hall et al., Plant Physiol. 107, 1379-1386 (1995); R. Sevenier et al., Nature Biotechnology 16, 843-846 (1998); and European Patent EP 0 723 393 B1. However, protoplasts isolated from sugar beet leaves vary in size and morphology, reflecting the high degree of cellular heterogeneity present within the source tissue at both physiological and cytogenetic levels. Accordingly, transformation techniques utilizing protoplasts requires the expert skill of laboratories that have developed particular methods, and the results are not easily reproducible.
At best, transformation techniques for sugarbeets have heretofore been very dependent on explant source, plant genotype and selection conditions used, and high efficiencies of transformation have been very difficult to achieve. See, e.g., K. Lindsey et al., J. Experimental Botany 41, 529-536 (1990); K. Lindsey et. al, “Transformation in Sugar Beet (Beta vulgaris L.),” in Biotechnology in Agriculture and Forestry, Vol. 23, Plant Protoplasts and Genetic Engineering IV (Y. P. S. Bajaj, Ed., Springer-Verlag, Berlin, 1993). Sugar beet is an important crop in the temperate climate region. Over 30% of the world's sugar consumption comes from sugar beet. There is thus a continuing need for a simple, high efficiency transformation method which may be applicable to beet and other plants that have heretofore been recalcitrant to transformation.
Other plants recalcitrant to transformation for which simple, high efficiency transformation methods are needed include the various species of squash. In one study, summer squash cultivars were regenerated via somatic embryogenesis using cotyledons excised from seeds. C. Gonsalves, HortScience 30, 1295-1297 (1995). However, the regeneration efficiency of this method was calculated as 0.3 plantlets per initial explant. Id. In other reports, the production of transgenic squash is reported, but the transformation procedures used to obtain the regenerated plants are not described or publicly available. See, e.g., D. Tricoli et al., Bio/Technology 13, 1458-1465, 1464 (1995).
Transformation methods using seedling (non-excised) shoot tips or excised shoot tips have been described. See U.S. Pat. No. 5,164,310 to Smith et al. (incorporated herein by reference in its entirety); P. Chee et al., Plant Physiol. 91, 1212-1218 (1989); F. J. L. Aragao et al., Int. J. Plant Sci. 158, 157-163 (1997); and P. Christou et al., Plant J. 8, 275-281 (1992). In these methods, shoot tips either remained attached to seedlings or germinating seeds at the time of transformation, or were excised from seedlings. In the latter case, only one shoot per excised shoot tip was ultimately produced.
Transformation methods using shoot tip-derived meristematic cultures have also been described. See, e.g., U.S. Pat. No. 5,767,368 to Zhong et al. (incorporated herein by reference in its entirety); H. Zhong et al., Plant Physiol. 110, 1097-1107 (1996); and S. Zhang et al., Plant Cell Reports 18, 959-966 (1999). However, these methods have been shown to be effective in only a handful of monocotyledonous plants (i.e., corn, barley and oat), and no transformed dicot plants have been successfully regenerated following transformation attempts using those methods. Furthermore, transformation of monocots using shoot-tip derived meristematic cultures, as described in the above references, was generally achieved by microprojectile bombardment (i.e., generally not mediated by Agrobacterium tumefaciens).